The endocrine pancreas is both the substrate for fundamental questions in developmental biology as well as the target of the disease diabetes mellitus, which affects millions of individuals worldwide. A detailed genetic and molecular understanding of pancreatic endocrine development will be essential if we are to be able to manipulate islet cell fate and numbers in vivo. Our emerging understanding of pancreatic development is one in which the initially unpatterned pancreatic epithelium is successively sub-divided into exocrine and endocrine compartments, followed by the differentiation of exocrine tissue into ductal and acinar cells and of the endocrine tissue into 4 types of islet cells, including the a and a cells that produce glucagon and insulin. Among the factors that dictate cell specification of the endocrine pancreas are two genes belonging to the Pax gene family, Pax4 and Pax6. Pax4 knockout mice lack beta cells, while Pax6 mutant Sey1Neu/Sey1Neu mice demonstrate reduced numbers of all 4 types of pancreatic endocrine cells. Mice lacking both Pax4 and Pax6 fail to develop any mature pancreatic endocrine cells. Thus, Pax6 is required for normal islet cell development, and other studies have indicated a role in the regulation of islet hormone transcription, suggesting an even broader role in pancreatic development. Our studies on Pax6 have identified a specific 450 bp enhancer element that is sufficient to confer Pax6 expression in early pancreatic islet cell progenitors. Molecular analysis has disclosed the identity of two factors, Meis4 (Prep1) and Pbx1, that appear to interact cooperatively to directly regulate the activity of this enhancer in vivo. In this proposal, we will follow up on these preliminary results via three Specific Aims. In Aim 1, we will further test the hypothesis that the transcription factors Meis4 and Pbx1 directly regulate the Pax6 pancreatic enhancer, and we will clarify the molecular mechanism by which they do so. In Aim 2, we will seek to identify additional transcription factors that regulate the Pax6 pancreatic enhancer. Lastly, in Aim 3, we will test whether, in adult animals, the Pax6 pancreatic enhancer recapitulates the reactivation of endogenous Pax6 that occurs during islet cell neogenesis, and whether Pax6 is sufficient to induce islet cell neogenesis. Collectively, these experiments should rigorously define key molecular interactions that regulate Pax6 expression in the developing endocrine pancreas and provide insight into the regulatory hierarchy controlling pancreatic development.